1. To identify key differentiation genes and characterize their molecular mechanisms of action A. Identification, Cloning and Characterization of CASZ1, the human homolog of the drosophila neural fate determination gene castor (dCas). We have identified, cloned and characterized CASZ1, the human homolog of the drosophila neural fate determination gene, dCas. We determined it mapped to chromosome 1p36.22, an allele was lost in both N-myc (NMA) amplified and non-NMA neuroblastoma cell lines and CASZ1 expression increased during retinoid differentiation. In drosophila, dCas is expressed exclusively at a late stage of neuroblast development that precedes the cessation of proliferation and the beginning of neuronal differentiation. The stage at which castor is expressed during drosophila development is analogous to later stages of human embryonic neural organogenesis, which implies that the human homologue- may have the same role as dCas in controlling cell fates within neuroblast cell lineages. The fact that this gene is homologous to a drosophila neural fate determination gene, is regulated during retinoid induced differentiation of NB cells and localizes to a chromosomal region of interest in NB (1p36) provided the rational to clone and characterize HCASZ1. Using in silico and standard cloning techniques, we identified two isoforms. Hcasz5 encodes a protein with 5 zinc fingers and belongs to the TFIIIA family of transcription proteins. There is a highly conserved para-ZnF sequence located upstream of each classic zinc finger, a feature that is identical to the zinc fingers in dCas. Hcasz11 is identical to hcasz5 for the first 1166 AA but contains an additional 6 TFIIIA class C2H2 zinc finger motifs that also contain the highly conserved para-ZnF sequence. As anticipated, both isoforms localize to the nucleus consistent with their putative function as transcriptional regulators. Both isoforms are highly expressed in human heart, skeletal muscle and fetal but not adult brain. Cell lines from NB tumors and rhabdomyosarcomas, a tumor of muscle origin, express to varying levels of the CASZ1 gene. CASZ1 gene expression was significantly increased after induction of differentiation in neuroblasts and C2C12 myoblasts, indicating that CASZ1 may play a role in controlling cell fates not only within neuroblast cell lineages but in myoblast cell lineages as well. B. MYCN regulation of gene expression in neuroblastoma cells Previously we had identified a number of siRNAs to target the MYCN gene. We have developed vector systems that can over-express and turn-off MYCN. These have been invaluable in our studies to identify MYCN targets involved in cell cycle regulation and differentiation. In a series of collaborative studies we have contributed to studies that identified that MYCN expression in tumors stimulates recruitment of natural killer cells to tumors. Our studies also contributed to the recent identification that the candidate tumor suppressor microRNA miR-34a directly regulates the MYCN gene. Both these studies have importance as they increase our understanding as to why NB tumors that have over-expression of MYCN have a more aggressive phenotype and patients have the worst overall survival. C. The Cancer Stem Cell hypothesis proposes that a rare cell in the tumor which is highly resistant to current therapies is responsible for repopulating the tumor while the progeny of this Cancer stem cell which are responsible for the bulk of the tumor are relatively more sensitive to the therapies. This theory would explain why tumors relapse. We developed a unique orthotopic model for neuroblastoma that enabled us to assess the tumor intitiating capacity of tumors. Contrary to the Cancer Stem Cell hypothesis we find that 1/10 cells in a primary culture of neuroblastoma cells and 1/2 cells from a neuroblastoma cell line is capable of initiating a tumor. This indicates that the almost every cell in a NB tumor has tumor initiating capacity.